1. Field of the Invention
The present invention relates to novel expression vectors which permit tight regulation of gene expression in eucaryotic cells. The invention also relates to methods for producing proteins and RNA molecules and methods for administering proteins and RNA molecules to a plant or animal.
2. Related Art
The ability to precisely control the expression of genes introduced into animal or human cells, or in whole organisms, will enable significant progress in many areas of biology and medicine. For instance, methods that allow the intentional manipulation of gene expression will facilitate the analysis of genes whose expression cannot be tolerated constitutively or at a certain stage of development. These methods will also be valuable for clinical applications such as gene therapy, where the expression of a therapeutic gene must be regulated in accordance with the needs of the patient.
To be of broad benefit, gene regulation techniques must allow for rapid, robust, precise and reversible induction of gene activity. As reviewed in Saez, E. et al., (Curr. Opin. Biotechnol. 8:608-616(1997)), an ideal system should fulfill the following requirements:    1. Specificity—The system must be indifferent to endogenous factors and activated only by exogenous stimuli.    2. Non-interference—The components of the system should not affect unintended cellular pathways.    3. Inducibility—In the inactive state, the basal activity of the system should be minimal, while in the active state high levels of gene expression should be rapidly inducible.    4. Bioavailability of the inducer—Inducing stimuli should rapidly penetrate to the site of interest.    5. Reversibility—Inducing stimuli should clear swiftly to allow the system to rapidly return to the inactive state.
Early methods for controlling gene expression in mammals were based on endogenous elements, such as cytokine response elements or heat-shock proteins. Due to a high level of basal expression in the uninduced state, and pleiotropic effects brought about by general inducing agents, these systems lacked the specificity required to regulate genes in mammalian cells and organisms.
More advance schemes have sought to avoid these problems by constructing switching mechanisms that rely on non-mammalian elements. The fundamental principle of these systems is based on the existence of a small molecule (the inducer) that modifies the activity of a synthetic transcription factor which regulates the expression of the target gene through a heterologous promoter Increased specificity is achieved by selecting inducers that do not affect mammalian physiology, and by assembling chimeric transactivators with minimal homology to natural transcription factors which do not interact with endogenous mammalian promoters.
The most common system currently in use for the regulation of gene expression is the tetracycline-based system (Gossen and Bujard, Proc. Natl. Acad Sci. USA 89:5547 (1992)). This system is based on the continuous expression of a fusion protein where the tetracycline repressor protein (tetR) is converted into an activator by fusion to the transcriptional activation domain of the VP16 protein. In the absence of tetracycline, this chimeric tetracycline transactivator (tTA) activates gene expression through binding to a multimer of the natural tetR binding site (tetO) placed upstream of a minimal promoter. In the presence of tetracycline, the tTA undergoes a conformational change that prevents it from binding to the tetO site, thereby arresting expression of the target gene. Because of its significant advantages over the existing approaches, the tTA system is highly useful for inducible gene expression and this system has been successfully used for the production of a number of proteins (Wimmel et al., Oncogene 9:995 (1994); Fruh et al., EMBO J. 13:3236 (1994); Yu et al., J. Virol. 70:4530 (1996)).
However, serious problems resulting from the toxicity of the tTA protein have been reported with the tTA system, and several cell types have been shown to be unable to tolerate expression of the tTA protein (Schocket et al., Proc. Natl. Acad. Sci. USA 92:6522 (1995); Howe et al., J Biol. Chem. 23:14168 (1995); Schocket and Schatz, Proc. Natl. Acad. Sci. USA 93:5173 (1996); Bohl et al., Nat. Med. 3:299 (1997)). While the toxicity of tTA in cultured cells encumbers the establishment of stable clones with proper tetracycline regulation, this tTA toxicity is a more significant problem in gene therapy and may prevent the use of the tTA system in gene therapy altogether.
A further problem of the tTA system is its notable degree of basal expression. Basal expression can result from the activation of the reporter constructs in the absence of bound transactivator, and/or the inability of tetracycline to completely quell tTA transactivation. High basal expression limits the inducibility of the system, and prevents the conductance of experiments with highly toxic proteins (Furth et al., Proc. Natl. Acad. Sci. USA 91:9302 (1994); Hennighausen et al., J. Cell. Biochem. 59:463 (1995) Kistner et al., Proc. Natl. Acad. Sci. USA 93:10933 (1996); Hoffmann et al., Nucleic Acids Res. 25:1078 (1997)).
In the case of stable clones or transgenic animals, some of this basal expression can be attributed to interference from chromosomal regions into which the foreign DNA integrates. While all inducible systems are equally susceptible to integration effects, it is possible that the basal activity of the tTA system is due to the fact that this system requires the constant presence of tetracycline to efficiently suppress transcription, something that may not always be attainable, particularly in vivo. Basal expression and the requirement that tetracycline be present to suppress gene expression are reasons why the tTA system is not used in gene therapy.
Two gene control systems based on components of mammalian steroid hormone receptors are known (Saez, E. et al., Curr. Opin. Biotechnol. 8:608-616 (1997)). Both combine a truncated form of the progesterone receptor hormone-binding domain with the yeast GAL4 DNA-binding moiety, and the transactivation domain of VP16 protein. The mutated progesterone receptor moiety fails to bind progesterone, but it retains the ability to bind the progesterone and glucocorticoid antagonist mifepristone (RU486), such that, in the presence of RU486, the fusion protein (called GVLP or TAXI) activates transcription through a multimer of the GAL4 DNA binding site placed upstream of a minimal promoter.
An important advantage of the systems described immediately above is that they appear to have more favorable kinetics than tetracycline approaches because lipophilic hormones are quickly metabolized and have short half-lives in vivo. Further, such hormones may also penetrate less accessible tissues more efficiently than tetracycline. However, the main disadvantage of the hormone receptor systems is their very high level of basal expression. In transient and stable transfections of various cell types, a high level a basal activity dampens the inducibility of theses approaches, resulting in induction ratios that are rarely over 20-fold (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180 (1994); Mangelsdorf et al., Cell 83:835 (1995); Wang et al., Nat. Biotech. 15:239 (1997)).
Another approach to regulating gene expression relies on a method of inducing protein dimerization derived from studies on the mechanism of action of immunosuppressive agents (Saez, E. et al., Curr. Opin. Biotechnol. 8:608-616 (1997)). Using a synthetic homodimer of FK506, a general strategy was devised to bring together any two peptides simply by endowing them with the domain of FKBP12 to which FK506 binds. Since immunosuppressive drugs, such as cyclosporin A or rapamycin must be used in this approach, the in vivo application of this protein dimerization approach is very limited.
All of the above mentioned strategies regulate expression by controlling the level of transcription of mRNA. Since this mRNA transcription mechanism is always influenced to some extent by the chromosomal region into which the foreign DNA is inserted, precise regulation fails due to the lack of control over the integration mechanism. Although techniques are available for the site-specific insertion of DNA by homologous recombination, insertion frequencies are far too low to allow this strategy to succeed for gene therapy on a general basis.
Another gene expression system is based on alphaviruses (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582 (1997)). Several members of the alphavirus family, Sindbis (Xiong, C. et al, Science 243:1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11:18-22 (1993)), SFV (Liljeström, P. & Garoff, H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N. L. et al., Virology 171:189-204 (1989)), have received considerable attention for the use as virus-based expression vectors for a variety of different proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582 (1997); Liljeström, P., Curr. Opin. Biotechnol. 5:495-500 (1994)).
Alphaviruses are positive stranded RNA viruses which replicate their genomic RNA entirely in the cytoplasm of the infected cell and without a DNA intermediate (Strauss, J. and Strauss, E., Microbiol. Rev. 58:491-562 (1994)). The concept that alphaviruses can be developed as expression vectors was first established nearly ten years ago (Xiong, C. et al., Science 243:1188-1191 (1989)). Since then, several improvements have made the use of these RNA replicons as expression vectors more practical (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582 (1997)).
DNA vectors have been developed for both Sindbis (Herweijer, H. et al., Hum. Gene Ther. 6:1495-1501 (1995); Dubensky, T. W. et al., J. Virol. 70:508-519 (1996)) and SFV (Berglund, P. et al., Trends Biotechnol. 14:130-134 (1996)). Eukaryotic promoters are introduced in these vectors upstream from the alphavirus replicase gene (consisting of the four non-structural protein genes (nsP1-4)) which are translated as one or two polyproteins which are then proteolytically cleaved (Strauss, J. and Strauss, E., Microbiol. Rev. 58:491-562 (1994)). DNA is transcribed to RNA from the recombinant eukaryotic promoter in the nucleus and transported to the cytoplasm, where the replicase catalyzes the replication of the alphavirus RNA molecule as during normal replication of the alphavirus RNA molecule (Strauss, J. and Strauss, E., Microbiol. Rev. 58:491-562 (1994)). Only transient expression of heterologous sequences has been possible until recently due to the cytopathogenicity of the alphavirus replicase (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582 (1997)).
About 20 years ago Weiss et al. (Weiss, B. et al., J. Virol. 33:463-474 (1980)) established a persistently infected culture of BHK cells. The mutation responsible for this phenotype has been recently identified (Dryga, S. A. et al., Virology 228:74-83 (1997)). Another mutation allowing the regulation of the mRNA transcription via temperature shifts was identified by Burge and Pfefferkorn (Burge, B. W. & Pfefferkorn, E. R., Virology 30:203-214 (1966)) and described in more detail by Xiong et al. (Xiong, C. et al., Science 243:1188-1191 (1989)).
Vectors containing alphaviral sequences have been developed which show promise for use in DNA immunizations (Hariharan, M. et al, J. Virol. 72:950-958 (1998)), ribozyme expression (Smith S. et al., J. Virol. 71:9713-9721 (1997)), and in vivo expression of heterologous proteins in mammalian tissues (Altman-Hamamdzic S. et al., Gene Ther. 4:815-822 (1997)).